The present invention relates to processes for preparing and stabilizing food-grade marine oils.
Marine oils have attracted substantial interest as a source of n-3 long-chain polyunsaturated fatty acids (LCPUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are of dietary significance. These LCPUFA contain 5 or 6 double bonds which render them prone to atmospheric oxidation accompanied by a fishy taste and smell. The increasing interest in LCPUFA has prompted research into methods of stabilizing fish oils against oxidation and off-flavor development
Refined marine oils are initially free from a fishy taste and smell but reversion through oxidation occurs rapidly. Many attempts have been made to stabilize such oils by the addition of different anti-oxidants or mixtures thereof. However, all these attempts have failed so far, cf. R. J. Hamilton et al., Journal of American Oil and Chemist""s Society (JAOCS), Vol. 75, no. 7, p. 813-822, (1998). Accordingly, there is a need for a process for stabilizing marine oils over a long period of time in a simple and economical way, where, even after a long period of storage, no fishy taste and smell occur.
Fully or partially refined marine oil which has been treated with silica and stabilized by incorporation in the thus-treated oil of a mixture of lecithin, ascorbyl palmitate, and alpha-tocopherol in accordance with the procedure described in EP 612 346 and its U.S. counterpart U.S. Pat. No. 5,855,944 (U.S. Pat. No. 5,855,944) reportedly displays excellent Rancimat stability and good application performance for health food supplements. In a particular embodiment of the procedure described in EP 612 346/U.S. Pat. No. 5,855,944, the filly or partially refined marine oil is treated with silica having a surface area greater than 500 m2/g, and the silica-treated oil is then subjected to a soft vacuum steam deodorization at a temperature between about 140xc2x0 C. and about 210xc2x0 C.; then lecithin, ascorbyl palitate and a tocopherol in the ratio of 6-3: 4-2: 8-4 are incorporated in the thus-treated oil, whereby the resulting stabilization is reported to last for several months. In dairy applications, such as yogurt and milk drinks, however, the so-treated and stabilized oil develops a strong fishy smell and taste.
Refined marine oil which has been treated with an adsorbent such as silica and stabilized with 0.1% deodorized rosemary extract (HERBALOX xe2x80x9cOxe2x80x9d, available from Kalsec, Inc. of Kalanmazoo, Mich.) and sage extract in a manner analogous to the procedure described in EP 340 635 reportedly has a hereby taste and smell which can be detected in food applications. This hereby taste and smell reportedly suppresses the taste and smell of fish. In dairy applications, the use of as little as 0.03% of HERBALOX xe2x80x9cOxe2x80x9d and sage extract in the marine oil results in a very strong hereby taste and smell which prevents the use of this oil in these applications.
Accordingly, the present invention provides a process for preparing and stabilizing food-grade marine oil which includes treating marine oil with silica optionally in the presence of carbon, vacuum steam deodorizing the marine oil at a temperature between about 140xc2x0 C. and about 210xc2x0 C. in the presence of 0.1-0.4% rosemary or sage extract, and, optionally, adding 0.01-0.03% ascorbyl palmitate and 0.05-0.2% mixed tocopherol to the marine oil after the deodorizing.
It has now surprisingly been found in accordance with the present invention that marine oil which has been treated with silica in accordance with the procedure described in EP 612 346/U.S. Pat. No. 5,855,944 can be stabilized over a long period of time without the occurrence of fishy taste and smell by vacuum steam deodorization at a temperature between about 140xc2x0 C. and about 210xc2x0 C., in the presence of 0.1-0.4% of deodorized rosemary or sage extract. The treating of the oil with silica may be by combining (i.e., adding or mixing the oil with silica). The treatment of the oil with silica can suitably be carried out in one embodiment by contacting the oil with the silica in a silica-filled column or stirred reactor vessel or a combination thereof Batch, semi-batch or continuous operation is feasible. Silica having a surface area of more than 500 m2/g is suitably used. It is preferred to carry out this treatment at about room temperature, although lower or, especially, higher temperatures may also be used if desired. Further, it is preferred to perform this embodiment under the atmosphere of an inert gas, especially nitrogen. The contact time between the oil and the silica can be varied within wide limits and can be a few seconds to several days. In this connection, the flow rate at which the oil passes through the silica in a column procedure will depend on factors such as the type and particle size of the chosen silica, the dimensions of the column and the like.
In another embodiment, an oil/solvent miscella is passed through a silica column The solvent is preferably a food grade approved apolar solvent, preferably a hydrocarbon and especially hexane. This embodiment can be carried out, for example, by dissolving the oil in the solvent to provide a solution containing about 20-60%, preferably 33%, oil by weight, passing the solution through silica in a column or stirred reactor vessel or a combination thereof using a ratio of miscella to silica of 20:1 to 5:1, preferably 15:1, (wt./wt.), filtering and then removing the solvent by distillation. Here again, the contact time between oil and the silica can be varied within wide limits and can be a few seconds to several days.
The treatment with silica described hereinbefore can also be carried out in the presence of carbon. The carbon which is used is preferably dried or substantially freed from water 2a before or during the process and, furthermore, preferably has a surface area of more than 1100 m2/g. Examples of suitable carbons are those available as NORIT, e.g. NORIT CA1, and similar activated carbons.
The fully refined marine oil used in the present invention is one which has been neutralized, bleached and deodorized in a conventional manner. The oil can be, for example, menhaden oil, herring oil, sardine oil anchovy oil, pilchard oil, tuna oil, shark oil, hake oil, etc., or a blend of two or more of these oils.
Factors associated with the fishy taste and smell of a marine oil are not well defined. In order to obtain more information as to which factors are responsible for the fishy taste and smell, 21 oil samples were analyzed in detail as shown and discussed below. Samples 1-10 used in these analytical proceedings are commercially available standard fish oils from suppliers throughout the world and are regarded as being xe2x80x9cagedxe2x80x9d because of the delays in refining them further in accordance with the procedure described in EP 612 346/U.S. Pat. No. 5,855,944, whereas samples 11-15 are refined fish oils where it is known that both the extraction and refining have been performed immediately after the fish have been caught or with minimum delay only. Samples 16-17 are oils of fungal origin Samples 18-21 have been produced from commercially available fish oils in accordance with the procedure described in EP 612 346/U.S. Pat. No. 5,855,944 in which, however, a special short path distillation step has been included at the start of the process to trap smell molecules for use as described below. The purpose to this wide trawl (diversified catch) is to have as representative a range as possible of refined oils containing EPA and DHA.
Table 1 records the influence of the acid value, the EPA and, respectively, DHA content, the color and the pro-oxidant iron and copper levels on sensory responses of a trained panel to the 21 oil samples described above.
The analysis for the determination of the EPA and DHA content and, respectively, the proxidant iron and copper levels were performed according to analytical methods known in the art. For determining the acid value, i.e., the number of milligrams of potassium hydroxide required to neutralize the free fatty acids in 1 gram of oil, the oil sample is titrated with 0.1N aqueous potassium hydroxide solution using a 1% phenolphthalein indicator. The size of the sample was determined as follows:
The color is determined by means of a Lovibond tintometer Model E AF 900 by matching the color of light transmitted through a specified depth of oil to the color of the light originating from the same source, transmitted through standard color slides. The results are expressed in terms of the red (R), yellow (Y), and blue (B) units to obtain the match and the size of the cell used. Taste and smell are sensorically evaluated by a trained panel of 12-15 persons. The panelists are asked to rank the samples in terms of perception of fishy taste and smell. A hedonic scale of 1 to 5 is used to express the extent of fishiness in which 1 represents no fishy taste or smell while 5 represents a very strong fishy taste or smell. The samples are coded using a three-digit code and 10-15 ml of each sample are submitted to the panel in a plastic beaker at 22xc2x0 C. The products are evaluated after processing after 4 weeks and, respectively, 12 weeks storage at a temperature of 22xc2x0 C. in aluminum containers.
Table 2 shows the effect of prima and secondary oxidation levels on the taste and smell of the same marine oils as in Table 1. Primary oxidation is measured as the peroxide value of the oils in milliequivalent (meq)/kg of oil. Secondary oxidation is measured in two ways: The first is by the reaction of unsaturated aldehydes in the oil with anisidine. The second is by the reaction of a alkenals and alkadienals in the oil with NN-dimethyl-p-phenylenediamine.
For determining the peroxide value, the oil is treated in a solution of acetic acid and chloroform with a solution of iodide, and subsequently the free iodine is titrated with a solution of sodium thiosulphate. The size of the sample was determined as follows:
The p-anisidine value as used herein is defined as 100 times the absorbence measured at 350 nm in a 1 cm cell of a solution containing 1.0 g of the oil in 100 ml of a mixture of hexane and a solution of p-anisidine in glacial acetic acid (0.025 g/100 ml of glacial acetic acid). The size of the sample was determined as follows:
The aldehyde values were determined based on a method described by K. Miyashita et al., JAOCS, Vol. 68 (1991), which discloses a process in which N,N-diethyl-p-phenylenediamine is reacted with aldehydes in the presence of acetic acid. The three aldehyde classes (alkanal, alkenal, and alkadienal) are determined by visible absorption at 400, 460 and 500 nm, respectively. The aldehyde values are expressed in mmole/kg.
Furthermore, the level of smell molecules in each of these oils has been measured by static headspace coupled to GC/MS (gas chromatograph/mass spectrometer). The oil to be measured (samples of 1 g each) is crimp sealed into a headspace vial (22 ml) in a nitrogen atmosphere and heated at 120xc2x0 C. for 15 minutes in a headspace autosampler. A measured of the headspace is automatically injected onto a GC/MS using a heated transfer line. chromatograph is used to separate the molecules, and the mass spectrometer is used to identify and quantify the separated molecules. The results obtained are shown in Table 3.
Table 1 shows that there is no correlation between the acid value, the EPA and DHA content, the color and pro-oxidant iron and copper levels, and the taste and smell of these marine oils.
Table 2 shows that the oxidation indicators are not capable of distinguishing oils with a good taste and smell from oils with a bad taste and smell.
Table 3 shows that static headspace cannot distinguish between good and bad tasting marine oils.
Tables 1-3 also show that marine oils which have been refined soon after the oil has been extracted from freshly caught fish do not exhibit better sensory response than oils which have been refined from aged crude fish oil. However, levels of secondary anisidine reactives and aldehydes are extremely low in these fresh oils. These results suggest that whatever is responsible in the marine oil for the fishy taste and smell is present at extremely low levels which are below the detection limits of static headspace GC/MS. The data also show that neither anisidine nor aldehyde measurements are very useful in predicting the sensory quality of the oil because they are too insensitive.
Tables 1-3 show sensory data for single cell oils which demonstrate that they too may become fishy in both taste and smell. Table 1 also shows that, when using specially refined oils, it is possible to produce marine oils with excellent taste and smell, but with quality parameters such as anisidine, peroxide, iron, copper, color and static headspace values which are not different from those of oils having poor taste and smell.
To understand the extent to which fishy taste and smell occur in marine oils, efforts have been made to identify and quantify the molecules responsible for the fishy taste and smell. Marine oils (1 kg each) rich in EPA and/or DHA which had a strong fishy smell were passed slowly through a short path distillation apparatus at 120xc2x0 C. and under reduced pressure (0.005 mbar). Two vacuum traps were connected in series, each cooled with liquid nitrogen, to collect the fishy volatiles which were removed by this process. These oils were then deodorized at 190xc2x0 C. and are designated in Tables 1-3 as samples 18-21. Even though their traditional quality parameters are no different from those of oils which are deemed to be fishy, they had little or no fishy taste The condensation products captured in the vacuum traps were dissolved in methyl tert butyl ether and subjected to the olfactory detector GC/MS to identify fishy molecules which had been removed by this process. In the olfactory detector GC/MS, the outlet stream from a gas chromatograph is split and routed to two different detectors. In the present case, the detectors used were the mass spectrometer and the human nose. Such a system allows peaks to be identified by the MS and assigned smell by an operator.
A number of very potent smell molecules were identified in the distillation products and are set forth in Table 4.
As can be seen from Table 3, only a few of the molecules listed in Table 4 could be identified by static headspace. Thus, a more sensitive method was required to remove headspace molecules from the oils. The detection limits for 2-octenal and 2,4-hexadienal, for example, were 940 ppb and 500 ppb, respectively. In order to improve the sensitivity of detection, the technique of dynamic headspace has been used. According to this technique, 2 g aliquots of oil were heated to 75xc2x0 C. in a water bath purged with helium (150 ml/min) through a Tekmar purge glass apparatus onto Perkin Elmer cartridges containing TENAX adsorbent (Enka Research Institute, Amheim). The dynamic headspace was measured by is GC/MS using a 30 m column of DB5-MS (1 xcexcm film thickness).
Table 5 below shows the taste panel response to a number of matures of marine oils and the dynamic headspace profile of a number of molecules. They have been identified by GC/MS and olfactory detector GC/MS. As can be seen, some of these molecules may be detected to single figure ppb levels, using dynamic headspace. The importance of the data in Table 5 is that they explain why the data in Tables 1-3 cannot possibly correlate with marine oil taste and smell, and they also demonstrate the very small amount of oxidation which is required before the oil deteriorates to an unacceptable quality in respect of taste and smell.
Table 6 below shows the excellent agreement between the level of 6 specially selected molecules in the headspace of the oils and the ranking by the taste panel using a multiple discriminant analysis (MDA). MDA is a statistical test for determining whether a given classification of cases into groups is a likely one. It will indicate whether the group assignment of a case is true or false. The final data are presented in a table with rows and columns corresponding to actual and estimated group membership, respectively. In the ambit of the present invention, the classification obtained from the sensorical evaluation by the taste panel was the taste factor. The MDA analysis was done through a statistical package called UNISTAT version 4.51.
The retention index of a compound is calculated from injections of C5-C15 saturated straight chain hydrocarbons under the same chromatographic conditions as the analysis of interest, and is similar to its retention time in that the longer it is retained on a GC column, the greater is its retention index/time. The use of the retention indices rather than retention times makes the information more rigorous and transferable, although the retention indices are still dependant on the column phase and chromatographic conditions, but still minimize instrument dependent variables.
In order for a peak on a GC trace to be accepted as having a certain identity, certain conditions must be met. The traditional condition with GC is that it should have the same retention index/time as an authentic standard. Of the 6 molecules listed, standards were obtained for 5 of them. Alternatively, mass spectra may be used as an additional tool to confirm peak identity.
Table 7 below shows the effect of increasing concentration of deodorized rosemary extract on the rancimat stability of a marine oil by adding it after deodorization.
Table 7 shows that between 0 and 4% addition of rosemary extract, the rancimat induction time and, thus, the rancimat stability of marine oil, increases with an increasing the amount of rosemary extract. Nevertheless, the use of rosemary extract as a stabilizer of marine oil after deodorization, is, even at the low amount of 0.2%, disadvantageous due to the powerful hereby smell of the commercial deodorized rosemary extract, particularly if it is put into dairy food applications. This makes it impossible to use the dose benefits shown in Table 7.
It has also been found in accordance with the present invention that adding the rosemary extract to the oil before deodorization removes the powerful smell without removing or destroying the anti-oxidant activity. The results of the relevant experiments are set forth in Tables 8 and 9.
Table 8 below shows a range of headspace molecules which describe the headspace of deodorized rosemary extracts at a concentration of 0.2% added to deodorized marine oil after deodorization and, respectively, 0.2% and 0.4% added before deodorization. In the latter case, two deodorization temperatures are given.
The relative values set forth in column 2 of Table 8 were derived from the analysis of marine oil with 0.2% HERBALOX xe2x80x9cOxe2x80x9d added after deodorizing. When the oils are deodorized it is necessary to have a concentration against which it is possible to measure removal of the headspace molecules. Therefore, the concentration of each compound found in the experiment in which the rosemary extract was added after removal was taken as 100%, and the effects of deodorizing were measured against this level.
Table 8 also shows that when a mineral oil to which 0.2% of rosemary oil has been added before deodorization, is deodorized at 150xc2x0 C. or 190xc2x0 C., virtually all of these spicy molecules are removed from the oil. With 0.4% addition, removal of most of the spicy is molecules is low; two of the main components, i.e. camphor and caryophyllene, are not completely removed.
The hereby smell in an oil deodorized at 150xc2x0 C. with 0.4% addition of rosemary extract before deodorization is still strong, whereas an oil with only 0.2% rosemary extract added does not have any hereby smell.
Table 9 below shows the effect on the anti-oxidant system of the following variables: the deodorization temperature, the anti-oxidant mixture, and the additional of rosemary before or after the deodorization.
As shown in Table 9, adding 0.2% rosemary extract to the marine oil without deodorizing increases the rancimat stability from 1.7 to 3.0.hours at 100xc2x0 C. The same or approximately the same rancimat stability is seen when the rosemary extract is added to the oil after deodorizing at 150xc2x0 C. and 190xc2x0 C. A slight increase in rancimat stability is observed when sage extract is added to the oil after deodorizing at 190xc2x0 C. If the rosemary extract is added to the oil before the deodorization at 150xc2x0 C., there is a slightly increased rancimat stability, but by deodorizing at 190xc2x0 C. in the presence of rosemary and sage extract, the rancimat stability of the oil is increased substantially to 4.1 and 3.4 hours, respectively. The addition of 0.02% ascorbyl palmitate and 0.1% mixed tocopherol after deodorization further enhances the rancimat stability of the oil. Thus, by deodorizing the oil at 190xc2x0 C. and adding 0.2% rosemary and sage extract, respectively, before the deodorization, followed by 0.02 % ascorbyl palmitate and 0.1% mixed tocopherol after the deodorization, it is possible to increase the rancimat stability of the oil from 1.7 to 6.2 and 5.3 hours, respectively.
The present invention is a process for the preparation and stabilization of food-grade marine oil by treating marine oil with silica in the presence or absence of carbon, vacuum steam deodorizing at a temperature between about 140xc2x0 C. and about 210xc2x0 C. in the presence of 0.1-0.4% rosemary or sage extract and, if desired, adding 0.01-0.03% ascorbyl palmitate and 0.05-0.2% mixed tocopherol. The invention further includes methods of using the oil thus obtained in food applications. A further object of the present invention is a method of determining the sensory quality of an unknown marine oil by measuring the dynamic headspace profile of the marine oil with regard to the 6 following compounds: (2)oheptenal (E)-2-hexenal, 1,5-(Z)-octadien-3-one, (E,E)-2,4-heptadienal, 3,6-nonadienal, and (E,Z)2,6-nonadienal; and evaluating the results obtained against the results of the oils set forth in Table 5 by multiple discriminant analysis.
In the present invention, the silica treatment is preferably performed in the presence of carbon. The preferred temperature for the deodorization step, which may be carried out in conventional equipment, is between about 150xc2x0 C. and about 190xc2x0 C., for example, about 190xc2x0 C. The preferred amount of deodorized rosemary or sage extract present during deodorization is about 0.2%. Furthermore, it is preferred to add after deodorization about 0.01-0.03%, preferably about 0.02%, of ascorbyl palmitate and about 0.05-0.2%, preferably about 0.1%, of mixed tocopherol. Any tocopherol can be used in this preferred embodiment, such as xcex1 and xcex3-tocopherols or a mixture of natural tocopherols.
The silica used in the present invention has been described in detail in EP 612 3461 / U.S. Pat. No. 5,855,944. This silica can be any conventional silica such as, for example, those available as TRISYL and TRISYL 300 (Grace), BRESORB (Akzo) or SD959, SD1027 and SORBSIL C60 (Crosfield). It is preferred to use a silica which is dried or substantially freed from water before or during the process, i.e. which preferably has a water content of up to about 2%, preferably up to about 1%. The drying of the silica can be achieved, for example, by heating at about 100xc2x0 C. for about 3 hours. Alternatively, the silica can be dried in heated oil under a vacuum or by azeotropic distillation. The carbon which can optionally be used has also been described in detail in EP 612 346/U.S. Pat. No. 5,855,944 and also above. All oils used in the examples were mixed with 5% silica and 2% activated carbon at 80xc2x0 C. and then filtered as described in EP 612 346/U.S. Pat. No. 5,855,944.